Systems and methods for combining hyperspectral images with color images

ABSTRACT

The invention is directed to methods and systems of hyperspectral and multispectral imaging of medical tissues. In particular, the invention is directed to new devices, tools and processes for the detection and evaluation of diseases and disorders such as, but not limited to diabetes and peripheral vascular disease and cancer, that incorporate hyperspectral or multispectral imaging.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/692,131 entitled “Hyperspectral Imaging of Angiogenesis,filed Mar. 27, 2007, which claims priority to U.S. ProvisionalApplication No. 60/785,977 entitled Hyperspectral Imaging ofAngiogenesis, filed Mar. 27, 2006, and is a continuation in part of U.S.application Ser. No. 11/689,783 entitled Hyperspectral Imaging inDiabetes and Peripheral Vascular Disease, filed Mar. 22, 2007, whichclaims priority to U.S. Provisional Patent Application No. 60/784,476entitled Combinations of Hyperspectral Imaging Methods with OtherEvaluation Methods, filed Mar. 22, 2006, and is a continuation in partof U.S. application Ser. No. 11/396,941 entitled Hyperspectral Imagingin Diabetes and Peripheral Vascular Disease filed Apr. 4, 2006, whichclaims priority to U.S. Provisional Application No. 60/667,677 entitledHyperspectral Imaging in Diabetes, filed Apr. 4, 2005, and U.S.Provisional Application No. 60/785,977 entitled Hyperspectral Imaging ofAngiogenesis, filed Mar. 27, 2006. U.S. patent application Ser. No.11/692,131 is also a continuation in part to U.S. application Ser. No.11/288,410 entitled Medical Hyperspectral Imaging for Evaluation ofTissue and Tumor filed Nov. 29, 2005, which claims priority to U.S.Provisional Application No. 60/631,135 entitled Hyperspectral Imaging inMedical Applications, filed Nov. 29, 2004, U.S. Provisional ApplicationNo. 60/667,678 entitled Hyperspectral Imaging in Breast Cancer, filed onApr. 4, 2005, and U.S. Provisional Application No. 60/732,146 entitledHyperspectral Analysis for the Detection of Lymphoma, filed Nov. 2,2005. All of these provisional and non-provisional applications arehereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The invention is directed to methods and systems of hyperspectral andmultispectral imaging of biological and medical tissues. In particular,the invention is directed to new devices, tools and processes for thedetection and evaluation of diseases and disorders such as diabetes andperipheral vascular disease that are amenable to diagnosis usinghyperspectral/multispectral imaging.

2. Background of the Invention

Diabetes afflicts an estimated 194 million people worldwide, affecting7.9% of Americans (over 21 million people) and 7.8% of Europeans.Between 85% and 95% of all diabetics suffer from Type 2 diabetes,although nearly 5 million people worldwide suffer from Type 1 diabetes,affecting an estimated 1.27 million people in Europe and another 1.04million people in the United States. Both Type 1 and Type 2 diabeticpatients are at higher risk for a wide array of complications includingheart disease, kidney disease (e.g. nephropathy), ocular diseases (e.g.glaucoma), and neuropathy and nerve damages to name a few². The feet ofdiabetic patients are at risk for a wide array of complications, whichare discussed below. Problems with the foot that affect the ambulatorynature of the patient are not only important from the standpoint ofphysical risk, but also convey an emotional risk as well, as theseproblems disrupt the fundamental independence of the patient by limitinghis or her ability to walk.

Peripheral arterial disease (PAD) affects primarily people older than55. There are currently 59.3 million Americans older than 55, and over12 million of them have symptomatic peripheral vascular disease. It isestimated that only 20% of all patients with PAD have been diagnosed atthis time. This represents a dramatically underpenetrated market.Although pharmacologic treatments for PAD have traditionally been poor,2.1 million nevertheless receive pharmacologic treatment for thesymptoms of PAD, and current diagnostic tests are not considered to bevery sensitive indicators of disease progression or response to therapy.Additionally, 443,000 patients undergo vascular procedures such asperipheral arterial bypass surgery (100,000) or peripheral angioplasty(343,000) annually and are candidates for pre and post surgical testing.One difficulty in diagnosing PAD is that in the general population, onlyabout 10% of persons with PAD experience classic symptoms ofintermittent claudication. About 40% of patients do not complain of legpain, while the remaining 50% have leg symptoms which differ fromclassic claudication.

Relying on medical history and physical examination alone isunsatisfactory. In one study, 44 percent of PAD diagnoses were falsepositive and 19 percent were false negative when medical history andphysical examination alone were used.³ For this reason, physicians havelooked for other means to help in providing diagnosis. As in the case ofdiabetic foot disease, current technologies have fallen short.Nonetheless, patients are frequently sent to peripheral vascularlaboratories for non-invasive studies. While the test results are knownto be inaccurate, these results do provide some additional informationto physicians for assistance in diagnosis or treatment decisions.

Another problem faced by physicians is disease of the peripheral veins.Venous occlusive disease due to incompetent valves in veins designed toprevent backflow and deep vein thrombosis results in venous congestionand eventually stasis ulcers. Approximately 70% of leg ulcers are due tovenous occlusion. Many of these ulcers are found at the medialmalleolus. The foot is generally swollen and the skin near the ulcersite is brownish in appearance.

Pathology

Diabetic feet are at risk for a wide range of pathologies, includingmicrocirculatory changes, peripheral vascular disease, ulceration,infection, deep tissue destruction and metabolic complications. Thedevelopment of an ulcer in the diabetic foot is commonly a result of abreak in the barrier between the dermis of the skin and the subcutaneousfat that cushions the foot during ambulation. This, in turn, can lead toincreased pressure on the dermis, resulting in tissue ischemia andeventual death, and ultimately result in an ulcer.⁴ There are a numberof factors that weigh heavily in the process of ulceration⁵-affectingdifferent aspects of the foot—that lead to a combination of effects thatgreatly increase the risk of ulceration:⁶

-   -   Neuropathy—Results in a loss of protective sensation in the        foot, exposing patients to undue, sudden or repetitive stress.        Can cause a lack of awareness of damage to the foot as it be        occurs and physical defects and deformities⁷ which lead to even        greater physical stresses on the foot. It can also lead to        increased risk of cracking and the development of fissures in        calluses, creating a potential entry for bacteria and increased        risk of infection.⁸    -   Microcirculatory Changes—Often seen in association with        hyperglycemic damage.⁹ Functional abnormalities occur at several        levels, including hyaline basement membrane thickening and        capillary leakage. On a histologic level, it is well known that        diabetes causes a thickening of the endothelial basement        membrane which in turn may lead to impaired endothelial cell        function.    -   Musculoskeletal Abnormalities—Include altered foot mechanics,        limited joint mobility, and bony deformities, and can lead to        harmful changes in biomechanics and gait. This increases        pressures associated with various regions of the foot.        Alteration or atrophy of fat pads from increased pressure can        lead to skin loss or callus, both of which increase the risk of        ulceration by two orders of magnitude.    -   Peripheral Vascular Disease—Caused by atherosclerotic        obstruction of large vessels resulting in arterial        insufficiency¹⁰ is common in the elderly populations and is yet        more common and severe in diabetics.¹¹ Diabetics may develop        atherosclerotic disease of large-sized and medium-sized        arteries, however, significant atherosclerotic disease of the        infrapopliteal segments is particularly common. The reason for        this is thought to result from a number of metabolic        abnormalities in diabetics, including high LDL and VLDL levels,        elevated plasma von Willebrand factor, inhibition of        prostacyclin synthesis, elevated plasma fibrinogen levels, and        increased platelet adhesiveness.    -   Venous Disease—Caused by incompetent valves controlling backflow        between the deep veins and the more superficial veins or        thrombosis of the deep veins. Venous occlusions are typically        observed in the elderly who typically presented with swollen        lower extremities and foot ulcers typically at the medial        malleolus.

Previous studies have shown that a foot ulcer precedes roughly 85% ofall lower extremity amputations in diabetic patients^(12, 13) and that15% of all diabetic patients will develop a foot ulcer during the courseof their lifetimes.¹⁴ More than 88,000 amputations performed annually ondiabetics,¹⁵ and roughly an additional 30,000 amputations are performedon nondiabetics, mostly related to peripheral vascular disease.Estimations have shown that between 2-6% of diabetic patients willdevelop a foot ulcer every year^(13, 16) and that the attributable costfor an adult male between 40 and 65 years old is over $27,000 (1995 USdollars) for the two years after diagnosis of the foot ulcer.¹⁶ Inconjunction with the increased total costs of care, Ramsey et al showedthat diabetic patients incurred more visits to the emergency room (morethan twice as many as control patients), more outpatient hospital visits(between 2× and 3× as many as control subjects) and more inpatienthospital days (between 3× and 4× as many as control patients) during thecourse of an average year.

Foot pathology is major source of morbidity among diabetics and is aleading cause of hospitalization. The infected and/or ischemic diabeticfoot ulcer accounts for about 25% of all hospital days among people withdiabetes, and the costs of foot disorder diagnosis and management areestimated at several billion dollars annually.^(16, 17)

Current Diagnostic Procedures

The first step in the assessment of the diabetic foot is the clinicalexamination^(18, 19). All patients with diabetes require a thoroughpedal examination at least once a year, even without signs ofneuropathy. Evaluation of the diabetic patient with peripheral vasculardisease should include a thorough medical history, vascular history,physical examination, neurologic evaluation for neuropathy and athorough vascular examination.²⁰

The next step in the work up of a patient with significant peripheralvascular or diabetic foot disease is non-invasive testing.²¹ Currentclinical practice can include ankle brachial index (ABI), transcutaneousoxygen measurements (TcPO2), pulse volume recordings (PVR) and laserDoppler flowmetry. All of these clinical assessments are highlysubjective with significant inter- and intra-observer variabilityespecially in longitudinal studies. None of these methods arediscriminatory for feet at risk, and none of them provide anyinformation about the spatial variability across the foot. Dopplerultrasound with B-mode realtime imaging is typically used to diagnosedeep vein thrombosis while photo and air plethysmography are used tomeasure volume refill rates as a means of locating and diagnosingvalvular insufficiency. Currently there is no method to accuratelyassess the predisposition to serious foot complications, to define thereal extent of disease or to track the efficacy of therapeutics overtime.

SUMMARY OF INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies, techniques, instrumentation anddesigns, and provides new tools and methods for detecting tissue at riskof developing into an ulcer, for detecting problems with diabetic footdisease, for assessing general tissue damage and metabolic state, andfor evaluating the potential for wounds to heal.

One embodiment of the invention is directed to a medical instrumentcomprising a first stage optic responsive to illumination of tissue, aspectral separator, one or more polarizers, an imaging sensor, adiagnostic processor, a filter control interface, a general purposeoperating module to assess the state of tissue in diabetic subjectsfollowing a set of instructions, and a calibrator. Preferably, theinstrument further comprises a second stage optic responsive toillumination of tissue. Preferably, the set of instructions comprisespreprocessing hyperspectral information, building a visual image,defining a region of interest in tissue, converting the visual imageinto units of optical density by taking a negative logarithm of eachdecimal base, decomposing a spectra for each pixel into severalindependent components, determining three planes for an RGB pseudo-colorimage, determining a sharpness factor plane, converting the RGBpseudo-color image to a hue-saturation-value/intensity image having aplane, adjusting the hue-saturation-value/intensity image plane with thesharpness factor plane, converting the hue-saturation-value/intensityimage back to the RGB pseudo-color image, removing outliers beyond astandard deviation and stretching image between 0 and 1, displaying theregion of interest in pseudo-colors; and characterizing a metabolicstate of the tissue of interest.

Preferably, the region of interest is one of a pixel, a specified regionor an entire field of view. Preferably, determining three planes for anRGB pseudo-color image comprises one or more characteristic features ofthe spectra, determining a sharpness factor plane comprises acombination of the images at different wavelengths, removing outliersbeyond a standard deviation comprises three standard deviations,displaying the region of interest in pseudo-colors comprises one ofperforming one in combination with a color photoimage of a subject, inaddition to a color photo image of a subject, and projecting onto thetissue of interest.

Preferably, defining the color intensity plane as apparent concentrationof one or a mathematical combination of oxygenated Hb, deoxygenated Hb,and total Hb, oxygen saturation, defining the color intensity plane asreflectance in blue-green-orange region, adjusting the hue saturationcomprises adjusting a color resolution of the pseudo-color imageaccording to quality of apparent concentration of one or a mathematicalcombination of oxygenated Hb, deoxygenated Hb, and total Hb, oxygensaturation, adjusting the hue saturation further comprises one or acombination of reducing resolution of hue and saturation color planes bybinning the image, resizing the image, and smoothing the image throughfiltering higher frequency components out, and further interpolating thesmoothed color planes on a grid of higher resolution intensity plane.

Another embodiment of the invention is directed to a method forassessing the state of tissue of a diabetic subject comprising,preprocessing hyperspectral information, building a visual image,defining a region of interest in tissue, converting the visual imageinto units of optical density by taking a negative logarithm of eachdecimal base, decomposing a spectra for each pixel into severalindependent components, determining three planes for an RGB pseudo-colorimage, determining a sharpness factor plane, converting the RGBpseudo-color image to a hue-saturation-value/intensity image having aplane, adjusting the hue-saturation-value/intensity image plane with thesharpness factor plane, converting the hue-saturation-value/intensityimage back to the RGB pseudo-color image, removing outliers beyond astandard deviation and stretching image between 0 and 1, displaying theregion of interest in pseudo-colors; and characterizing a metabolicstate of the tissue of interest.

Preferably, the region of interest is one of a pixel, a specified regionor an entire field of view. Preferably, determining three planes for anRGB pseudo-color image comprises one or more characteristic features ofthe spectra, determining a sharpness factor plane comprises acombination of the images at different wavelengths, removing outliersbeyond a standard deviation comprises three standard deviations,displaying the region of interest in pseudo-colors comprises one ofperforming one in combination with a color photoimage of a subject, inaddition to a color photo image of a subject, and projecting onto thetissue of interest.

Preferably, defining the color intensity plane as apparent concentrationof one or a mathematical combination of oxygenated Hb, deoxygenated Hb,and total Hb, oxygen saturation, defining the color intensity plane asreflectance in blue-green-orange region, adjusting the hue saturationcomprises adjusting a color resolution of the pseudo-color imageaccording to quality of apparent concentration of one or a mathematicalcombination of oxygenated Hb, deoxygenated Hb, and total Hb, oxygensaturation, adjusting the hue saturation further comprises one or acombination of reducing resolution of hue and saturation color planes bybinning the image, resizing the image, and smoothing the image throughfiltering higher frequency components out, and further interpolating thesmoothed color planes on a grid of higher resolution intensity plane.

Another embodiment is directed to quantifying an increase in thevasculature around a wound, and can be used for comparisons to adjacenttissue. Embodiments of this invention can be used to quantify anincrease in vasculature as the result of a proangiogenic agent.Proangiogenic agents include, but are not limited to, vascularendothelial growth factors (VEGF), epidermal growth factor (EGF), tumornecrosis factor (TNF-α), interleukin-1α, and substance P. Otherembodiments quantify a decrease in vasculature as a result of anantiangiogenic agent. Antiangiogenic agents include, but are not limitedto, angiostatin, interferon-α, metalloproteinase inhibitors, and otherangiogenesis inhibitor drugs approved by the FDA. Other embodiments areused to quantify enhanced wound healing due to a proangiogenic agent.Preferably, enhanced wound healing is quantified due to a proangiogenicagent in diabetics. More preferably, embodiments are used to quantifyenhanced wound healing in diabetic foot ulcers due to a proangiogenicagent. Other embodiments are used to quantify delayed cancer growth dueto an antiangiogenic agent. Other embodiments are directed toquantifying a reduction in cancer size due to an antiangiogenic agent.Other embodiments are used to quantify a decrease in cancer growth dueto an antiangiogenic agent. Other embodiments are used to quantifyenhanced wound healing due to negative pressure wound therapy. Otherembodiments are directed to quantifying enhanced wound healing due tohyperbaric therapy.

Another embodiment is directed to automatic image processing/targetrecognition to highlight regions, tissues, or issues of interest.Another embodiment is directed to projecting an image into the field ofview of the operator of an apparatus of this invention in such a way asto provide further useful information than simply viewing the targettissue unaided would provide. Other embodiments of this invention aredirected to viewing tissues with an MHSI (multispectral/hyperspectralimaging) device. Other embodiments are directed to determining thestatus of a wound in absolute terms, as well as with respect to othertissues. Other embodiments are directed to quantifying the physiologicstates of tissue, or of tissue-like compounds.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Block diagram depicting a portable hyperspectral imagingapparatus.

FIG. 2: Basic specifications of the MHSI system.

FIG. 3: OxyHb and DeoxyHb HSV/I color chart. Schematic representation ofthe MHSI display (left) showing the interplay between the oxyHb anddeoxyHb coefficients and describing some of the potential physiologicalconsequences of values of the MHSI. In one embodiment, tissuesdetermined to have high oxyhemoglobin and low deoxyhemoglobin levels(upper left-hand quadrant of FIG. 3) are displayed in a color locatedproximal to a first terminal color (e.g., purple) along a color scaleand are faded. These tissues are provided high oxygen delivery and havelow oxygen extraction. The oxygen delivery in these tissues exceeds thetissue oxygen demand. These are healthy tissues having the lowest riskfor ulceration and the highest probability of healing. In oneembodiment, tissues determined to have high oxyhemoglobin and highdeoxyhemoglobin levels (upper right-hand quadrant of FIG. 3) aredisplayed in a color located proximal to a first terminal color (e.g.,purple) along a color scale and are bright. These tissues are providedhigh oxygen delivery and have high oxygen extraction. The balance ofoxygenated blood in these tissues reflects high perfusion and highmetabolic rates. These tissues are at lower risk for ulceration and havea probable likelihood of healing. In one embodiment, tissues determinedto have low oxyhemoglobin and high deoxyhemoglobin levels (lowerright-hand quadrant of FIG. 3) are displayed in a color located proximalto a second terminal color (e.g., brown) along a color scale and arebright. These tissues are provided low oxygen delivery and have highoxygen extraction. The oxygen demand in these tissues exceeds the oxygendelivery. These tissues are at risk for ulceration. In one embodiment,tissues determined to have low oxyhemoglobin and low deoxyhemoglobinlevels (lower left-hand quadrant of FIG. 3) are displayed in a colorlocated proximal to a second terminal color (e.g., brown) along a colorscale and are faded. These tissues are provided low oxygen delivery andhave low oxygen extraction, indicating the lowest perfusion. The oxygendelivery in these tissues exceeds is very low. These tissues have thehighest risk of ulceration.

FIG. 4: Representative data from dorsal surface of foot showingindividual oxyHb and deoxyHb values and how they can be used to evaluateregions of the tissue.

FIG. 5: Representative data from tissue showing sensitivity of MHSI todrug-induced changes in the vasculature. (left to right) Visible imageof foot surface post iontophoresis (IP), representative spectra pre andpost iontophoresis with Acetylcholine (IP) showing greater oxyHb levelsafter IP. Images of increased oxyHb coefficient ring where IP occurred,image of deoxyHb, showing little change post IP.

FIG. 6: Representative data from an ulcer located on the sole (ulcer 1)and dorsal surface (ulcer 2) of the foot.

FIG. 7: MHSI information from the soles and dorsal surfaces of fourpatients. Each row of images represents data from one patient. The twocolumns on the left represent data from the soles of the feet, while thecolumns on the right represent data from the dorsal surfaces of thefeet.

FIG. 8: MHSI image of diabetic foot ulcer with 200 segment radialprofile.

FIG. 9: MHSI of wounds during healing. The 50-micron resolution imagesof a rabbit's ear taken with MHSI (Medical Hyperspectral Imaging) system(HyperMed, Inc.) over 10 days period. Reconstructed from MHSI data,shows a part of the observed area 50-by-40 mm, recorded at the baselineon day 1. The black rings denote location of a future wound—puncture.

FIG. 10: Obtained as a result of hyperspectral processing, showsdistribution of the oxygenated (oxy) and deoxygenated (deoxy) hemoglobinin the underlying tissue at the same time. The color hue representsapparent oxy concentrations, whereas color saturation (from fade tobright) represents apparent deoxy concentrations. Both, oxy and deoxy,vary predominantly between 40 and 90 mhsi units (colorbar to the right).The series of images to the right show change in a region of interest17-by-17 mm (black box in a) and b)) over 10 days following the puncturewound initiated at day 1. At day 2, the oxy concentrations increasedsignificantly in the area as far as 10 mm away from the wound border. Byday 5, the increase in oxygenation became more local (purple area“shrunken” to about 5 mm) and new microvasculature formed to “feed” thearea in need (red fork-like vessels in the right top corners appearingin the images for days 5 and 10). By the 10th day, the area of increasedoxy has not changed much, but the peak in oxy amplitude decreased,suggesting a period of steady healing.

DESCRIPTION OF THE INVENTION

Background of Hyperspectral Imaging

HSI or hyperspectral imaging is a novel method of “imaging spectroscopy”that generates a “gradient map” of a region of interest based on localchemical composition. HSI has been used in satellite investigation ofsuspected chemical weapons production areas²², geological features²³,and the condition of agricultural fields²⁴ and has recently been appliedto the investigation of physiologic and pathologic changes in livingtissue in animal and human studies to provide information as to thehealth or disease of tissue that is otherwise unavailable.²⁵ MHSI formedical applications (MHSI) has been shown to accurately predictviability and survival of tissue deprived of adequate perfusion, and todifferentiate diseased (e.g. tumor) and ischemic tissue from normaltissue.²⁷

Spectroscopy is used in medicine to monitor metabolic status in avariety of tissues. One of the most common spectroscopic applications isin pulse oximetry, which utilizes the different oxyhemoglobin {oxyHb)and deoxyhemoglobin (deoxyHb) absorption bands to estimate arterialhemoglobin oxygen saturation.²⁸ One of the drawbacks of these systems isthat they provide no information about the spatial distribution orheterogeneity of the data. In addition, these systems report the ratioof oxyHb and deoxyHb together thereby losing diagnostic information thatcan be garnered by evaluating the state of the individual components.Such spatial information for the individual components and the ratio isprovided by HSI, which is considered a method of “imaging spectroscopy”,where the multi-dimensional {spatial & spectral) data are represented inwhat is called a “hypercube.” The spectrum of reflected light isacquired for each pixel in a region, and each such spectrum is subjectedto standard analysis. This allows the creation of an image based on themetabolic state of the region of interest (ROI).

In vivo, MHSI has been used to demonstrate otherwise unobserved changesin pathophysiology. Specific studies have evaluated the macroscopicdistribution of skin oxygen saturation,²⁷ the in-situ detection of tumorduring breast cancer resection in the rat,²⁷ the determination of tissueviability following plastic surgery & burns,^(30, 31) claudication andfoot ulcers in diabetic patients,³²⁻³⁷, and applications to shock andlower body negative pressure (LBNP) in pigs and humans,respectively.³⁸⁻⁴⁰ In a skin pedicle flap model in the rat, tissue thathas insufficient oxygenation to remain viable is readily apparent fromlocal oxygen saturation maps calculated from hyperspectral imagesacquired immediately following surgery; by contrast, clinical signs ofimpending necrosis do not become apparent for 12 hours after surgery.⁴¹

Non-invasive measurements of oxygen or blood flow have been demonstratedpreviously, with investigators using thermometry,⁴² point diffusereflectance spectroscopy^(43, 44) and laser Doppler imaging.⁴⁵ Sheffieldet al, have also reviewed laser Doppler and TcPO₂ measurements and theirspecific applications to wound healing.⁴⁶ While other techniques havebeen utilized in both the research lab and the clinic and have theadvantage of a longer experience base, MHSI is superior to othertechnologies and can provide predictive information on the onset andoutcomes of diabetic foot ulcers, venous stasis ulcers and peripheralvascular disease.

Because MHSI has the ability to show anatomically relevant informationthat is useful in the assessment of local, regional and systemicdisease. This is important in the assessment of people with diabetesand/or peripheral vascular disease. MHSI shows the oxygen delivery andoxygen extraction of each pixel in the image collected, These imageswith pixels ranging from 20 microns to 120 microns have been useful inseveral ways. In the case of systemic disease, MHSI shows the effects onthe microcirculation of systemic diabetes, smoking, a variety ofmedications such as all of the classes of antihypertensives (ACEinhibitors, ARBs, Beta blockers, Peripheral arterial and arteriolardilators), vasodilators (such as nitroglycerine, quinine, morphine),vasoconstrictors (including coffee, tobacco, pseudephedrine, Ritalin,epinephrine, levophedrine, neosynepherine), state of hydration, state ofcardiac function (baseline, exercise, congestive heart failure),systemic infection or sepsis as well as other viral or bacterialinfections and parasitic diseases. The size of the pixels used isimportant in that it is smaller than the spacing of the perforatingarterioles (˜0.8 mm)⁴⁷ of the dermis and therefore permits thevisualization of the distribution of mottling or other patternsassociated with the anatomy of the microcirculation and its responses.In the case of the use of MHSI for regional assessment, in addition tothe above systemic effects at play, the image delivers information aboutthe oxygen delivery and oxygen extraction for a particular region as itis influenced by blood flow through the larger vessels of that region ofthe body. For example an image of the top of the foot reflects both thesystemic microvascular status and the status of the large(macrovascular) vessels supplying the leg. This can reflectatherosclerotic or other blockage of the vessel, potential injury to thevessel with narrowing, or spasm of some of the smaller vessels. It canalso reflect other regionalized processes such as neuropathy or venousocclusion or compromise or stasis. In the case of local disease MHSIshows the actual effect of the combination of systemic, regional andlocal effects on small pieces of tissue. This combines the effects ofsystemic and regional effects described above with the effects of localinfluences on the tissue including pressure, neuropathy, localized smallvessel occlusion, localized trauma or wounding, pressure sore,inflammation, and wound healing. Angiogenesis during wound healing isreadily monitored with MHSI.

Wounds other than on the foot can be similarly assessed, such as sacraldecubiti, other areas of pressure necrosis, prosthesis stumps, skin flaptissue before, after or during surgery, areas of tissue breakdown aftersurgery, and burn injuries. In preferred embodiments of this invention,wounds that are assessed by this invention's imaging methods includewounds due to acute injuries such as lacerations, burns, bruises, woundsfrom high impact traumas, fractures, abrasions, bone dislocations,transfusion-related acute injuries, etc. Current optical methods forevaluating tissues for the conditions described above include:

-   -   Laser Doppler (LD)—In early iontophoresis experiments as well as        recent efforts both LD and MHSI data were collected, and some        changes in our images (total hemoglobin) are primarily a        consequence of changes in perfusion which was roughly correlated        to LD. However, important other changes in MHSI images that        report specifically O₂ extraction and tissue metabolism (O₂Sat)        are not related to perfusion or LD readings per-se. Superior        spatial resolution with MHSI, and O₂ extraction information adds        highly important clinical information.    -   Transcutaneous PO₂ (TcPO₂)—TcPO₂ data collected in subjects with        peripheral vascular disease and ischemia study as well as in        patients with diabetes both with and without foot ulcers. TcPO₂        measurements appeared cumbersome, lengthy (˜20-30 minutes),        highly operator dependant, and carried data only from skin        directly under the probe (with little ability to distinguish the        spatial characteristics of the ischemic area). While TcPO₂ has        been shown to carry statistically significant information in        terms of quantifying tissue at risk for ulceration,⁴⁸ TcPO₂ was        not encouraging as a useful clinical device.

Non-imaging techniques—Techniques such as near-infrared absorptionspectroscopy (NIRS) or TcPO₂, rely on measurements at a single point intissue which may not accurately reflect overall tissue condition orprovide anatomically relevant data, and probe placement on the skin canalter blood flow and cannot deliver accurate information in the area ofan ulcer or directly surrounding it. Because MHSI is truly remotesensing, data are acquired at a distance, eliminating probe placementerrors and allowing the investigation of the wound itself, which sometechniques can not accomplish due to infection risk.

In short, analysis of the present invention supports the followingconclusions:

-   -   1. Level of oxygenated hemoglobin in the tissue of arms and feet        of diabetic subjects is lower than the level of oxygenated        hemoglobin in the skin of control subjects. This is a        statistically significant result with separation between        diabetics and controls.³⁶    -   2. Oxyhemoglobin in the arms and feet of ulcerated subjects is        lower than oxyhemoglobin in diabetics without the ulceration.        The strong signal suggests ability to distinguish diabetics at        lower and high risk.    -   3. Oxygen saturation level in the skin of arms and feet of        diabetics is lower than oxygen saturation in the skin of        controls. This is at a statistically significant level allowing        separation between diabetics and controls.    -   4. MHSI quantitatively assesses different areas of tissue        metabolism on both dorsal and plantar foot surfaces of any        curvature.    -   5. MHSI evaluates state of tissue as a function of distance away        from ulcer to assess the viability of surrounding tissue, and        evaluate the degree of risk of further ulceration.    -   6. MHSI can be classified with a 4-quadrant system to determine        the metabolic state of tissue using oxygen delivery and oxygen        extraction: low/low, low/high, high/high, and high/low. This        metric is used in distinguishing healthy tissue from ulcerated,        or from a tissue at risk of ulceration.    -   7. MHSI is a unique visualization method that produces an image        that combines spatial information from three independent        parameters characterizing tissue: oxygenated and deoxygenated        hemoglobin concentrations and light absorption.    -   8. MHSI evaluates skin metabolism at high resolution of 20-120        microns per image pixel.    -   9. Specific MHSI regions associated with the margins of the        ulcer correlate to inflammation (and/or infection).    -   10. Areas of decreased MHSI indicate tissue at risk for        non-healing, ulcer extension, or primary ulceration.    -   11. MHSI differentiates between regions of tissue associated        with a present foot ulcer on the basis of biomarkers such as        oxyHb and deoxyHb coefficients.    -   12. MHSI evaluates temporal changes in oxygen delivery and        extraction to particular areas, both, on local and systemic        scale. The trend in the change of oxyHb and deoxyHb are used to        predict healing status of a wound/ulcer as well as progression        of diabetic complications.    -   13. Specific results from MHSI are indicative of inflamed        tissue.    -   14. MHSI examines tissue for gross features that may be        indicative of global risks of complications, such as poor        perfusion or the inability of the microcirculation to react and        compensate in tissue.    -   15. MHSI has potential in diagnosing global microcirculatory        insufficiencies and impacting on other complications of diabetes        associated with the microvasculature besides foot ulcers.

MHSI is superior to other modalities for assessing the healing potentialof tissue adjacent to ulcers. MHSI provides more direct measurements ofoxyHb and deoxyHb activities of the affected tissue. Hence, thediscrimination is not markedly improved by adding iontophoresis resultsto refine prediction as is required for Laser Doppler to do so. MHSI hassignificant advantages over laser Doppler and TcPO₂ measurements.Whereas MHSI is able to deliver spatially relevant data with highspatial resolution, TcPO₂ delivers only single point data. Laser Dopplerdata has poor spatial resolution and is frequently reported as a singlemean numerical value across the region of interest.

The major clinical advantage of hyperspectral imaging is the delivery ofmetabolic information derived from the tissue's spectral properties inan easily interpretable image format with high spatial resolution. This2-D information allows gradients in biomarker levels to be assessedspatially. Multiple images taken over time allow the gradient to bemeasured temporally. This adds new dimensions to the assessment ofulceration risk and tissue healing in that it will allow the physicianto target therapy and care to specific at risk areas much earlier thanpreviously possible. The reporting of biomarkers such as oxyHb anddeoxyHb levels in tissue individually and in an image format wherespatial distributions can be assessed has not been done before.Typically the two numbers are combined in a ratio and reported aspercent hemoglobin oxygen saturation (O₂Sat). MHSI has the clearpotential to be developed into a cost effective, easy to use, turn-keycamera-based metabolic sensor given the availability and relatively lowprice of components.

Surprisingly, MHSI information according to this invention can be usedto predict the onset of foot ulcers before there are clinicalindications, and provides early detection, diagnosis, and quantificationof progression of microcirculatory complications such as neuropathy indiabetic patients. For patients with foot ulcers, MHSI technology canevaluate the ulcer and surrounding area to predict whether that willheal or require surgical intervention. The present invention alsoprovides MHSI that is useful in the prediction and monitoring ofperipheral venous disease including venous ulcers.

There are many advantages to using MHSI. Not only does MHSI provideanatomically relevant spectral information, its use of spectral data ofreflected electro-magnetic radiation (ultraviolet—UV, visible, nearinfrared—NIR, and infrared—IR) provides detailed tissue information.Since different types of tissue reflect, absorb and scatter lightdifferently, in theory the hyperspectral cubes contain enoughinformation to differentiate between tissue types and conditions. MHSIis more robust than conventional analyses since it is based on a fewgeneral properties of the spectral profiles (slope, offset, water,oxyHb, deoxyHb, and its ratio) and is therefore flexible with respect tospectral coverage and not sensitive to a particular light wavelength.MHSI is faster than conventional analyses because it uses fast imageprocessing techniques that allow superposition of absorbance,scattering, and oxygenation information in one pseudo-color image.Visible MHSI is useful because it clearly depicts oxyHb and deoxyHbwhich are important, physiologically relevant biomarkers in a spatiallyrelevant fashion. Similarly, NIR shows water, oxyHb and deoxyHb.

The simplicity of the presented false color images representingdistribution of various chemical species, either singly or incombination (such as ratioed), or in other more sophisticated imageprocessing techniques allow for the display of results in real tonear-real time. Another advantage of MHSI is easy interpretation. Colorchanges show the different tissue types or condition, but thedistinction is not a yes/no type. MHSI color scheme allows the surgeonor podiatrist to differentiate between different tissue types andstates. In addition, the color and the shape of structures depictdifferent composition and level of viability of the tissue. The data isthen represented in a developed MHSI standard format. OxyHb and deoxyHbare presented in a format similar to a blood pressure reading that iseasy for physicians to understand. Additionally, a tissue oxygensaturation value denoted as S_(HSI)O₂ is also provided.

MHSI main purposes include 1) expand human capabilities beyond theordinary array of senses; 2) expand the human brain capabilities bypre-analyzing the spectral characteristics of the observable subject; 3)perform these tasks with real or near-real time data acquisition. Insummary, the aim of MHSI is to facilitate the diagnosis and assessmentof the metabolic state of tissue.

Results of analysis have to be presented in an easily accessible andinterpretable form. MHSI delivers results in an intuitive form bypairing MHSI pseudo-color image with a high quality color picturecomposed from the same hyperspectral data. Identification and assessmentof a region of interest (ROI) is easily achieved by flipping betweencolor and MHSI images, and zooming onto the ROI. The images can be seenon a computer screen or projector, and/or stored and transported as anyother digital information, and/or printed out. The MHSI image preservesthe high resolution of the hyperspectral imager thereby allowing furtherimprovement with upgraded hardware.

Additionally, MHSI transcribes vast 3D spectral information sets intoone image preserving biological complexity via millions of color shades.The particular color and distinct shape of features in the pseudo-colorimage allow discrimination between tissue types such as ulcers, callus,intact skin, hematoma, and superficial blood vessels.

Initially, the algorithm presents oxyHb, deoxyHb and S_(HSI)O₂ to theuser to conclude characteristics of the tissue including, but notlimited to, discerning whether the tissue is healing or whether it is ata high risk of ulceration. In another embodiment, a particular colorcode contains adequate information for diagnosis and is presented assuch. In one iteration, MHSI by itself is not a definite decision makingalgorithm; it is a tool that a medical professional can use in order togive a confident diagnosis. In another iteration, MHSI contains adecision making algorithm that provides the physician with a diagnosis.

Due to the complexity of the biological system, medical personnel desireas much information as possible in order to make the most-reliablediagnosis. MHSI provides currently unavailable information to thedoctor, preferably to be used in conjunction with other clinicalassessments to provide an accurate diagnosis. MHSI provides images forfurther analysis by the user. As more information is gathered, aspectral library is preferably compiled to allow MHSI to be a truediagnostic device.

MHSI is preferably used to quantify medical therapies in order tomeasure the effectiveness of new therapeutic agents or procedures. Forexample, in wound healing studies, a typical subject population can bebroken down into one of three groups: those that will heal independentof therapy, those that will not heal independent of therapy, and theborderline cases that may benefit from the therapy. MHSI preferably isused to select borderline subjects for these studies where the treatmentif effective most likely benefits the subject. MHSI is used to quantifywound progression or prevention in order to identify new therapeuticagents and to develop individual therapeutic regiments depending onsubject response.

One embodiment of the invention is directed to a medical instrumentcomprising a first-stage optic responsive to illumination of a tissue, aspectral separator, one or more polarizers, an imaging sensor, adiagnostic processor, a filter control interface, and a general-purposeoperating module (FIG. 1). Preferably, the spectral separator isoptically responsive to the first-stage optic and has a control input,the polarizer filters a plurality of light beams into a plane ofpolarization before entering the imaging sensor, the imaging sensor isoptically responsive to the spectral separator and has an image dataoutput, the diagnostic processor comprises an image acquisitioninterface with an input responsive to the imaging sensor and one or morediagnostic protocol modules wherein each diagnostic protocol modulecontains a set of instructions for operating the spectral separator andfor operating the filter control interface, the filter control interfacecomprises a control output provided to the control input of the spectralseparator, which directs the spectral separator independently of theillumination to receive one or more wavelengths of the illumination toprovide multispectral or hyperspectral information as determined by theset of instructions provided by the one or more diagnostic protocolmodule, and the general-purpose operating module performs filtering andacquiring steps one or more times depending on the set of instructionsprovided by the one or more diagnostic protocol modules.

The instrument may also comprise a second-stage optic responsive toillumination of the tissue. Preferably, the one or more wavelengths ofillumination are one or a combination of UV, visible, NIR, and IR. Inpreferred embodiments, the multispectral or hyperspectral informationdetermines one or more of the metabolic state of tissue to assess areasat high risk of developing into a foot ulcer or other wounded tissue toassess the potential of an ulcer or the tissue to heal. Preferredembodiments include multispectral or hyperspectral information gatheredremotely and noninvasively. Alternatively, an imaging system could beaffixed to a wounded area to track its progress over time. Such a systemcould be attached to or embedded in a dressing, skin covering or adevice used to impact wound healing or maintain tissue integrity such asa vacuum suction system or a bed upon which a patient is lying or ashoe, boot or offloading device.

Another embodiment is directed to the set of instructions comprising:preprocessing the hyperspectral information, building a visual image,defining a region of interest of the tissue, converting allhyperspectral image intensities into units of optical density by takinga negative logarithm of each decimal base, decomposing a spectra foreach pixel into several independent components, determining three planesfor an RGB pseudo-color image, determining a sharpness factor plane,converting the RGB pseudo-color image to ahue-saturation-value/intensity (HSV/I) image having a plane, scaling thehue-saturation-value/intensity image plane with the sharpness factorplane, converting the hue-saturation-value/intensity image back to theRGB pseudo-color image, removing outliers beyond a standard deviationand stretching image between 0 and 1, displaying the region of interestin pseudo-colors; and characterizing a metabolic state of the tissue ofinterest.

The region of interest may be a pixel, a group of pixels in aprespecified region of a prespecified shape or a handoutlined shape oran entire field of view. Preferably, determining the three planes for anRGB pseudo-color image comprises one or more characteristic features ofthe spectra. Preferably, determining a sharpness factor plane comprisesa combination of the images at different wavelengths, preferably bytaking a ratio of a yellow plane in the range of about 550-580 nm to agreen plane in the range of about 495-525 nm, or by taking a combinationof oxyHb and deoxyHb spectral components, or by taking a ratio between awavelength in the red region in the range 615-710 nm and a wavelength inthe yellow region in the range of about 550-580 nm or in the orangeregion in the range of about 580-615 nm. Preferably, outliers areremoved beyond a standard deviation, preferably three standarddeviations. The region of interest is displayed in pseudo-colors,performed with one of in combination with a color photo image of asubject, or in addition to a color photo image of a subject, or byprojecting the pseudo-color image onto the observed surface.

Another embodiment of the invention is directed to a method forevaluating DFU or area of tissue at risk comprising preprocessing thehyperspectral information, building a visual image, defining a region ofinterest of the tissue, converting all hyperspectral image intensitiesinto units of optical density by taking a negative logarithm of eachdecimal base, decomposing a spectra for each pixel into severalindependent components, determining three planes for an RGB pseudo-colorimage, determining a sharpness factor plane, converting the RGBpseudo-color image to a hue-saturation-value/intensity (HSV/I) imagehaving a plane, scaling the hue-saturation-value/intensity image planewith the sharpness factor plane, converting thehue-saturation-value/intensity image back to the RGB pseudo-color image,removing outliers beyond a standard deviation and stretching imagebetween 0 and 1, displaying the region of interest in pseudo-colors, andcharacterizing a metabolic state of the tissue of interest.

Another embodiment is directed to a medical instrument comprising animage projector 81, an illumination source, a remote control device 82and a real-time data processing package 85. Such a system could projectthe colorized or other kind of image with relevant information back ontothe tissue from which it was taken to assist the physician in diagnosisand treatment such as wound debridement. Alternatively, information canbe transmitted to the physician using multiple means, such as a heads-updisplay.

Another embodiment is intended to help tell the doctor level ofamputation, safety of debriding tissue, likelihood for tissue to heal,selection and monitoring of specific therapy including topicalpharmaceuticals, skin-like coverings, vacuum suction apparatus, systemicpharmaceuticals, adequacy of surgical, stenting or atherectomyprocedure, extension of infection vs inflammation of tissue to assist intherapy, identification of organism responsible for local or systemicinfection.

Yet another embodiment can give information about tissue hydration andpotentially information about oxyHb and deoxyHb from deeper tissue usingNIR wavelengths. These can be used as a stand alone device or as pairedwith the more standard Visible wavelength MHSI device as shown in FIG.2.

Yet another embodiment can derive and present information from changesseen radiating from an area of wounded, ulcerated or otherwise abnormaltissue or from any change in tissue characteristics over a distance. A“gradient map” thus produced can be used to generate a diagnosis,predict the capability of the tissue to heal, define a level foramputation, define the infection vs inflammation, define areas ofischemia, define areas of tissue at risk for ulceration etc.

Another embodiment can involve dividing the region of interest intoradial segments, pie like segments or a combination of the two or intosquares or other geometric shapes and using these segments to compareand contrast different regions of tissue in the same field of view or ascompared to a similar field of view on the contralateral extremity or onanother part of the body (such as the forearm, the upper leg, etc.). Theradial segments can also be compared to similar locations at differenttime points to demonstrate change over time in response to differenttherapeutic interventions, changes in tissue physiology, either local,regional or systemic due either to progression or remission of diseaseor of the effects of topical or systemic medications or therapies.

Such measurements can be used to evaluate wound healing, tissueregeneration, angiogenesis, vasculogenesis, arteriogenesis, infection,inflammation, microvascular disease or alterations, or other changes intissue characteristics or physiology associated with the implementationof negative pressure (vacuum suction applied to the wound), hyperbarictherapy, grafting of autologuous, heterograft, xenograft or biologicalor synthetic skin substituetes, administration of topical agentsincluding antibiotics, cleansers, growth factors, surgical intervention,angioplasty, stenting, atherectomy, laser therapy, vasodilator therapy,offloading, compression, effects of pressure due to orthotic orprosthetic, effects of electromagnetic, acupuncture, massage, infrared,vibration or other therapies.

Such measurements can be used to quantify an increase in the vasculaturearound a wound, and can be used for comparisons to adjacent tissue.Embodiments of this invention can be used to quantify an increase invasculature as the result of a proangiogenic agent. Proangiogenic agentsinclude, but are not limited to, vascular endothelial growth factors(VEGF), epidermal growth factor (EGF), tumor necrosis factor (TNF-α),interleukin-1α, and substance P. Other embodiments quantify a decreasein vasculature as a result of an antiangiogenic agent. Antiangiogenicagents include, but are not limited to, angiostatin, interferon-α,metalloproteinase inhibitors, and other angiogenesis inhibitor drugsapproved by the FDA. Other embodiments are used to quantify enhancedwound healing due to a proangiogenic agent. Preferably, enhanced woundhealing is quantified due to a proangiogenic agent in diabetics. Morepreferably, embodiments are used to quantify enhanced wound healing indiabetic foot ulcers due to a proangiogenic agent. Other embodiments areused to quantify delayed wound healing due to an antiangiogenic agent.Other embodiments are used to quantify a decrease in cancer growth dueto an antiangiogenic agent. Other embodiments are used to quantifyenhanced wound healing due to negative pressure wound therapy. Otherembodiments are directed to quantifying enhanced wound healing due tohyperbaric therapy.

Such measurements can be considered as biomarkers representing tissueoxygen delivery and oxygen extraction, tissue oxygenation, tissueperfusion, tissue metabolism or other characteristics correlated withMHSI measurements.

Such measurements can be used in association with the implementation ofhyperbaric therapy delivered to assist in the healing of ulceration indiabetic or other foot ulceration, or other wounds in other parts of thebody. In the case of hyperbaric oxygen therapy, the tissue can bemonitored before and at specified intervals during therapy orcontinuously during therapy to determine when the tissue has beenadequately modified (oxygenated) by the therapy or that there has beensufficient change in tissue metabolism as described by the MHSImeasurements of oxyHb, deoxy Hb or other measured parameters or whetherno benefit is being delivered. MHSI can be used to determine theappropriate duration of HBO therapy during a given session and as towhether sufficient benefit has been delivered from a course of therapythat it can safely be discontinued and that the wound will then belikely to heal with more standard methods.

MHSI can be used to determine the capability of tissue to heal afterdebridement and hence the relative safety of pursuing such an approach.Similarly, MHSI can be used to help determine the lowest level ofamputation that can be performed with successful healing. Similarly MHSIcan be used to determine whether elective surgery to the foot, lowerextremity or other body part where evaluation and or quantitation ofperfusion, oxygenation, or tissue metabolism would assist indetermination of the safety of undertaking such a procedure or thelocation in which to direct such a procedure. MHSI can be utilizedbefore debridement, amputation or other surgery to make thisdetermination or during debridement, amputation or other surgery tobetter assess tissue to improve surgical outcomes.

Such measurements can be used for the determination of which patients orwhich wounds are likely to improve with any of the above mentionedtherapies, which patients or wounds or portions of wounds are healing orworsening, when a given therapy is sufficient (this could be during orimmediately after application of a therapy such as hyperbaric therapy ora debridement or a particular cleansing or pharmaceutical regimen orafter a longer course of several days of therapy such as a vacuumtherapy. MHS criteria can be used to determine when a tissue will accepta skin graft or benefit from an allograft or other skin replacement.

Systemic or Regional Disease

One embodiment uses a single system that employs light wavelengthsranging from the UV through the far infrared portions of theelectromagnetic spectrum, as well as either side of this range as newtechnologies are developed allowing for use of a greater portion of thisspectrum (e.g. UV, visible, the near infrared, short wave infrared, midinfrared or far infrared portion of the electromagnetic spectrum).Another embodiment uses a system that uses one or more wavelengths frommore than one of these wavelength regimes. One such system usingwavelengths from more than one of these wavelength groupings is shown infigure two. In other embodiments, a single sensor could be used tocollect light from more than one wavelength regime.

A portable hyperspectral imaging apparatus according to an embodiment ofthe invention is depicted in FIG. 1. Portable apparatus 10 weighs lessthan 100 pounds, preferably less than 25 pounds, and more preferablyless than 10 pounds. Preferably, the portable apparatus may be batteryoperated, have some other form of portable power source or morepreferably, may have a connector adapted to connect to an existing powersource.

Portable apparatus 10 comprises an optical acquisition system 36 and adiagnostic processor 38. Optical acquisition system 36 comprises meansto acquire broadband data, visible data, ultraviolet data, infrareddata, hyperspectral data, or any combination thereof. In a preferredembodiment, optical acquiring means comprises a first-stage imagingoptic 40, a spectral separator 42, a second-stage optic 44, and animaging sensor 46. Alternatively, optical acquiring means may be anyacquisition system suited for acquiring broadband data, visible data,ultraviolet data, infrared data, hyperspectral data, or any combinationthereof. Preferably, one or more polarizers 41, 43 are included in theacquisition system to compile the light into a plane of polarizationbefore entering the imaging sensor. Preferably, a calibrator is alsoincluded in the system.

If the spectral separator 42 does not internally polarize the light, thefirst polarizer 43 is placed anywhere in the optical path, preferably infront of the receiving camera 46. The second polarizer 41 is placed infront of illuminating lights 20 such that the incident lightpolarization is controlled. The incident light is crossed polarized withthe light recorded by the camera 46 to reduce specular reflection orpolarization at different angles to vary intensity of the reflectedlight recorded by the camera.

The illumination is provided by the remote light(s) 20, various sourcestailored or adapted to the need of the instrument, preferably positionedaround the light receiving opening of the system, or otherwise placed toafford optimal performance. The light can be a circular array of focusedLED lights that emit light at the particular wavelengths (or ranges)that are used in the processing algorithm, or in the ranges ofwavelengths (e.g., visible and/or near-infrared). The circulararrangement of the light sources provides even illumination that reducesshadowing. The light wavelength selectivity reduces effect of theobservation on the observing subject. The configuration may also varydepending on the particular needs and operation of the system.

Although the preferred embodiment describes the system as portable, anon-portable system may also be utilized. Preferably, an optical head ismounted to the wall of the examination room, more preferably, anoverhead light structure is located in the operating room, or morepreferably, the system has a portable table with an observational windowoverlooking the operating site.

The preferred embodiment may also be used as part of another instrument.For example, as an adjunct to an endoscope.

The first-stage optic receives light collected from a tissue samplethrough a polarizer and focuses the light onto the surface of thespectral separator. Preferably, the spectral separator is a liquidcrystal tunable filter (LCTF). LCTF 42 is a programmable filter thatsequentially provides light from selected wavelength bands with small(for example, 7-10 nm) bandwidth from the light collected from thesample. Second-stage optic 44 receives the narrow band of light passingthrough the spectral separator and focuses the light onto the imagesensor 46. The image sensor is preferably, although not necessarily, atwo-dimensional array sensor, such as a charge-coupled device array(CCD) or CMOS, which delivers an image signal to the diagnosticprocessor 38.

Diagnostic processor 38 includes an image acquisition interface 50, thathas an input responsive to an output of the image sensor 46 and anoutput provided to a general-purpose operating module 54. Thegeneral-purpose operating module includes routines that perform imageprocessing, and that operates and controls the various parts of thesystem. The general-purpose operating module also controls the lightsource(s) (e.g. LED array) allowing for switching on and off duringmeasurement as required by the algorithm. The general-purpose operatingmodule has control output provided to a filter control interface 52,which in turn has an output provided to the spectral separator 42. Thegeneral-purpose operating module also interacts with a number ofdiagnostic protocol modules 56A, 56B, . . . 54N, and has an outputprovided to a video display. The diagnostic process includes specialpurpose hardware, general-purpose hardware with special-purposesoftware, or a combination of the two. The diagnostic processor alsoincludes an input device 58, which is operatively connected to thegeneral-purpose operating module. A storage device 60 and printer 62also are operatively connected to the general-purpose operating module.

In operation, a portable or semi-portable apparatus is employed withinline of site (or with optical access) of the object or area of interest,e.g., diabetic foot with or without an ulcer, or general area ofinterest. An operator begins by selecting a diagnostic protocol moduleusing the input device. Each diagnostic protocol module is adapted todetect particular tissue characteristics of the target. The diagnosticmodule could be specific for diabetes, for peripheral vascular disease,for venous stasis disease or for a combination of these disease states.As another example, a screening protocol for feet without ulcers or apotential for healing protocol for feet with ulcers. In an alternativeembodiment, the apparatus may contain only one diagnostic module adaptedfor general medical diagnosis.

Diagnostic processor 38 responds to the operator's input by obtaining aseries of transfer functions and an image processing protocol and animage processing protocol from the selected diagnostic protocol module56. The diagnostic processor provides the filtering transfer functionsto the spectral separator 42 via its filter control interface 52 andthen instructs the image acquisition interface 50 to acquire and storethe resulting filtered image from the image sensor 46. Thegeneral-purpose operating module 54 repeats these filtering andacquiring steps one or more times, depending on the number of filtertransfer functions stored in the selected diagnostic protocol module.The filtering transfer functions can represent bandpass, multiplebandpass, or other filter characteristics and can include wavelengths inpreferably the UV, preferably the visible, preferably the NIR andpreferably, the IR electromagnetic spectrum.

In a preferred embodiment, the light source delivering light to thetarget of interest can be filtered as opposed to the returned lightcollected by the detector. Thus, a tunable source delivers theinformation. Alternatively, both a tunable source and a tunable detectormay be utilized. Such tuning takes the form of LCTF, acousto-opticaltunable filter (AOTF), filter wheels, matched filters, diffractiongratings or other spectral separators. The light source may be atungsten halogen or xenon lamp, but is preferably a light emitting diode(LED).

The unique cool illumination provided by the LED prevents overheating ofskin which may result in poor imaging resolution. Preferably, the LEDprovides sufficient light while producing no other physical orphysiologic effects such as, for example, minimal or no increase in skintemperature. This lighting system in combination with the polarizerallows adequate illumination while preventing surface glare frominternal organs and overheating of skin. In certain embodiments,illumination can arise from any source meeting the needs of the devicesuch as, for example, more passive sources such as room light or fromsunlight.

Once the image acquisition interface 50 has stored images for all of theimage planes specified by the diagnostic protocol chosen by theoperator, the image acquisition interface begins processing these imageplanes based on the image processing protocol from the selecteddiagnostic protocol module 56N. Processing operations can includegeneral image processing of combined images, such as comparing therelative amplitude of the collected light at different wavelengths,adding amplitudes of the collected light at different wavelengths, orcomputing other combinations of signals corresponding to the acquiredplanes. The computed image is displayed on the display 12. Otherpreferred embodiments include storing the computed image in the storagedevice 60 or printing the computed image out on printer 62.

In a preferred embodiment, a calibrator is included in the system.Calibrator has an area colored with a pattern of two (or more) colors.To optimize use of the calibrator for this particular application whereoxyHb and deoxyHb are important components of the solution, colors arechosen that have a distinct absorption band in the wavelength rangesimilar to oxyHb and deoxyHb—preferably in the range 500-600 nm. Thecolors are placed into a pattern, preferably, a checker-board pattern,where 1 out of 4 squares has color1, and 3 out of 4 squares have color2.Thus, approximately 25% of the squares are color1 and 75% of the squaresare color2. The system takes a hypercube being slightly out offocus—that provides blurring of colors into each pixel. From the spectrafor each pixel, a linear composition of two spectra: one from color1 andanother from color2 are observed. The recorded spectra are decomposed ina manner similar to a system that decomposes skin spectra into oxyHb &deoxyHb components. However, in this instance it takes pure color1 andcolor2 spectra from library instead of oxyHb & deoxyHb. Validcalibration reports concentrations of 75% for color2 and 25% for color1.Results are similar to skin analysis, where the output is approximately90% of oxyHb and 10% of deoxyHb. Other embodiments include but are notlimited to, changes to the pattern, the color concentration & intensity,and the number of colors.

In summary, the calibrator simulates the way the biological mixture(oxyHb+deoxyHb) is observed by using “optical” mixture via combinationof pattern (with known spatial concentrations) and analog blurring(defocusing—for speed. Defocusing can also be done in the softwarethrough the use of computational filters) in such a way as to ensurethat the entire MHSI system is functioning correctly and accurately.

If the correct result is obtained, confirmation of the lightingdistribution and collection throughput, and the wavelength accuracy ofthe system given confidence in the spectra (wavelengths and intensity)that are being collected are provided. This provides additionalassurance that the data recorded off the patient is acceptable.

In another preferred embodiment, diagnostic protocol modules 56, printer62, display 12, or any combination thereof, may be omitted from portabledevice 10. In this embodiment, acquired images are stored in storagedevice 60 during the medical procedure. At a later time, these imagesare transferred via a communications link to a second device or computerlocated at a remote location, for example, hospital medical records, forbackup or reviewing at a later time. This second device can have theomitted diagnostic protocol modules, printer, display, or anycombination thereof. In another embodiment, the stored images aretransferred from portable device 10, located in the clinic, via acommunications link to a remote second device in real time.

In a preferred embodiment the system has facility to project real-timehyperspectral data onto the operation field, region of interest, orviewing window positioned above the operating site through use of aHeads Up Display or other suitable technique allowing the user tooverlay the image in a useful manner. Also, the hyperspectral data canbe displayed completely separately for remote guidance (i.e. on a wallscreen for a group of people to review in real time, or post procedure).The projected information has precise one-to-one mapping to theilluminated surface (e.g. wound, operating surface, tissue) and providesthe user with necessary information in efficient and non-distractiveway. When projected onto an overhang viewing window, the images(real-color and/or pseudo-color) can be zoomed in/out to providevariable magnification. This subsystem consists of the followingelements: 1) image projector 81 with field-of view precisely co-alignedwith the field-of view of the hyperspectral imager, 2) miniature remotecontrol device 82 which allows the surgeon or podiatrist to switch theprojected image on and off without turning from the site of debridementand change highlight structure and/or translucency on the projectedimage to improve visibility of the features of interest as well asprojected image brightness and intensity, 3) a real-time data processingpackage 85 which constructs a projected image based on hyperspectraldata and operator/surgeon input, 4) optional viewing window 84positioned above the operating site that is translucent for realobservation or opaque for projecting pseudo-color solution or higherresolution images.

The MHSI system consists of three functional modules—a Spectral Imager(SI), supporting Controller and Power Module (CPM) and Control and DataAcquisition Computer (CDAC). The MHSI also includes a thermometer thatremotely measures the temperature at the tissue surface. The SpectralImager is mounted on suspension arm which neutralizes device weight andallows for easy positioning and focusing of the instrument. Thesuspension arm is attached to wheeled cart which supports CPM and CDACas well. This configuration is very mobile and permits wide range ofdevice spatial and directional motions.

FIG. 2 shows the preferred system specifications along with a diagram ofour focusing methodology and the optical design of the Spectral Imager.In this embodiment, a liquid crystal tunable filters (LCTF's) was usedas the wavelength selector and are coupled to complementary metal oxidesemiconductor (CMOS) imaging sensors. Fitted with macro lenses and thepositional light focusing system described below, the system has apreferred working focal length of roughly 1 to 2 feet.

A major issue in the collection of hyperspectral imaging data is theposition and focusing of the instrument. While our Spectral ImagingModule is positioned on a ball joint that allows free rotation andvirtually any angle of incidence to the patient, it is imperative thatthere be a system in place for targeting the image to a particular spoton the tissue and ensuring that the instrument will be at the properdistance from the tissue to achieve optimal focus. Positioning andfocusing with our system are facilitated by two mirrored collimatedlight beams or lasers that cross precisely at the instrument focal plane(FIG. 2), and so bringing the spectral imaging module into positionwhere the two light spots overlap on the tissue to insure optimal focus.

To achieve precisely calibrated images, the system may use a speciallydesigned calibration pad placed at the focal plane of the system andmeasured prior to each patient measurement. The calibration pad includesa diffusely reflective surface to quantify the intensity of theillumination at each wavelength and color bars to validate wavelengthaccuracy of the system. Calibration data measured at a preset time suchas during maintenance calibrations can be stored and compared to witheach use to decide whether the system is within specifications andshould proceed to patient measurements.

To achieve precise co-registration between the hyperspectral image andthe operating surface, the system may use a fiducial label or targetplaced in the field of view which the image registration module can useto perform a self-alignment procedure before or during the operation asnecessary.

Devices of the present invention allow for the creation and uniqueidentification of patterns in data that highlight the information ofinterest. The data sets in this case may be discrete images, eachtightly bounded in spectra that can then be analyzed. This is analogousto looking at a scene through various colored lenses, each filtering outall but a particular color, and then a recombining of these images intosomething new. Such techniques as false color analysis (assigning newcolors to an image that don't represent the true color but are anartifact designed to improve the image analysis by a human) are alsoapplicable. Optionally, optics can be modified to provide a zoomfunction, or to transition from a micro environment to a macroenvironment and a macro environment to a micro environment. Further,commercially available features can be added to provide real-time ornear real-time functioning. Data analysis can be enhanced bytriangulation with two or more optical acquisition systems. Polarizationmay be used as desired to enhance signatures for various targets.

In addition to having the ability to gather data, the present inventionalso encompasses the ability to combine the data in various mannersincluding vision fusion, summation, subtraction and other, more complexprocesses whereby certain unique signatures for information of interestcan be defined so that background data and imagery can be removed,thereby highlighting features or information of interest. This can alsobe combined with automated ways of noting or highlighting items, areasor information of interest in the display of the information.

The hyperspectrally resolved image in the present invention is comprisedof a plurality of spectral bands. Each spectral band is adjacent toanother forming a continuous set. Preferably, each spectral band havinga bandwidth of less than 50 nm, more preferably less than 30 nm, morepreferably less than 20 nm, more preferably, from about 20-40 nm, morepreferably, from about 20-30 nm, more preferably, from about 10-20 nm,more preferably from about 10-15 nm, and more preferably from about 5-12nm.

It is clear to one skilled in the art that there are many uses for amedical hyperspectral imager (MHSI) according to the invention. The MHSIoffers the advantages of performing the functions for such uses faster,more economically, and with less equipment and infrastructure/logisticstail than other conventional techniques. Many similar examples can beascertained by one of ordinary skill in the art from this disclosure forcircumstances where medical personal relies on their visual analysis ofthe biological system. The MHSI acts like “magic glasses” to help humanto see inside and beyond.

Algorithm Description

The embodiment of diabetes algorithm involves the following steps:

-   -   1. Preprocess the MHSI data. Preferably, by removing background        radiation by subtracting the calibrated background radiation        from each newly acquired image while accounting for uneven light        distribution by dividing each image by the reflectance        calibrator image and registering images across a hyperspectral        cube.    -   2. Build a color-photo-quality visual image. Preferably, by        concatenating three planes from the hyperspectral cube at the        wavelengths that approximately correspond to red (preferably in        the range of about 580-800 nm, more preferably in the range of        about 600-700 nm, more preferably in the range of about 625-675        nm and more preferably at about 650 nm), green (preferably in        the range of about 480-580 nm, more preferably in the range of        about 500-550 nm, more preferably in the range of about 505-515        nm, and more preferably at about 510 nm), and blue (preferably        in the range of about 350-490 nm, more preferably in the range        of about 400-480 nm, more preferably in the range of about        450-475 nm, and more preferably at about 470 nm) color along the        third dimension to be scaled for RGB image.    -   3. Define a region of interest (ROI), preferably, where the        solution is to be calculated unless the entire field of view to        be analyzed.    -   4. Convert all hyperspectral image intensities into units of        optical density. Preferably, by taking the negative logarithm of        the decimal base. FIG. 2 shows examples of spectra taking from        single pixels at different tissue sites within an image. Tissue        sites include connective tissues, oxygenated tissues, muscle,        tumor, and blood.    -   5. Decompose the spectra for each pixel (or ROI averaged across        several pixels). Preferably, decompose into several independent        components, more preferably, two of which are oxyhemoglobin and        deoxyhemoglobin.    -   6. Determine three planes for pseudo-color image. Preferably,        define the color hue plane as apparent concentration of        oxygenated Hb, or deoxygenated Hb, or their mathematical        combination, e.g. total Hb, oxygen saturation, etc. Preferably,        define the color saturation plane as apparent concentration of        oxygenated Hb, or deoxygenated Hb, or their mathematical        combination, e.g. total Hb, oxygen saturation, etc. Preferably,        define the color intensity (value) plane as reflectance in        blue-green-orange region (preferably in the range of light at        about 450-580 nm).    -   7. Adjust the color resolution of the pseudo-color image        according to quality of apparent concentration of oxygenated Hb,        or deoxygenated Hb, or their mathematical combination, e.g.        total Hb, oxygen saturation, etc. Preferably, reduce resolution        of hue, and saturation color planes by binning the image (e.g.        by 2, 3, 4, etc. pixels), or/and by resizing the image, or/and        by smoothing the image through filtering higher frequency        components out. Interpolate the smoothed color planes on the        grid of higher resolution intensity (value) plane.    -   8. Convert hue-saturation-value/intensity (HSV/I) image to        red-green-blue (RGB) image.    -   9. Remove outliers in the resulting image, defining an outlier        as color intensity deviating from a typical range beyond certain        number of standard deviations, preferably three. Stretch the        resulting image to fill entire color intensity range, e.g.        between 0 and 1 for a double precision image.    -   10. Display ROI in pseudo-colors, preferably, in combination        with the color photo image of the subject, or preferably, in        addition to the color photo image of the subject, or more        preferably, by projecting the pseudo-color image onto the        observed surface. Additional information can be conveyed through        images portraying the individual coefficients from oxyHb,        deoxyHb, slope and offset coefficients, or any linear or        nonlinear combination such as the oxyhemoglobin to        deoxyhemoglobin ratio.    -   11. Characterize the metabolic state of the tissue of interest        (e.g. risk for ulceration, potential to heal). Preferably, by        using the saturation and/or intensity of the assigned color and        provide a qualitative color scale bar.

As is clear to a person of ordinary skill in the art, one or more of theabove steps in the algorithm can be performed in a different order oreliminated entirely and still produce adequate and desired results.Preferably, the set of instructions includes only the steps ofpreprocessing the hyperspectral information, building a visual image,using the entire field of view, converting all hyperspectral imageintensities into units of optical density by taking a negative logarithmof each decimal base, and characterizing a metabolic state of the tissueof interest. More preferably, the set of instructions comprisespreprocessing the hyperspectral information, defining a region ofinterest of the tissue, and characterizing a state of the tissue ofinterest.

Another preferred embodiment entails reducing the hyperspectral data inthe spectral dimension into a small set of physiologic parametersinvolves resolving the spectral images into several linearly independentimages (e.g. oxyhemoglobin, deoxyhemoglobin, an offset coefficientencompassing multiple scattering (MS) properties and a slopecoefficient) in the visible regime. Another embodiment determines fourimages (e.g. oxyhemoglobin, deoxyhemoglobin, offset/scatteringcoefficient, and water absorption) in the near infrared region of thespectrum. As an example for the visible region of the spectrum, linearregression fit coefficients c1, c2, c3 and c4 will be calculated forreference oxy-Hb, deoxy-Hb, and MS spectra, respectively, for eachspectrum (Sij) in an image cube:{right arrow over (S)} _(ij) =∥c ₁{right arrow over (OxyHb)}+c ₂{rightarrow over (DeoxyHb)}+c ₃{right arrow over (Offset+c ₄Slope)}∥₂Individual images of the oxyhemoglobin and deoxyhemoglobin components,the slope and offset or any combination, linear or nonlinear, of theseterms, for example the oxy- to deoxyhemoglobin ratio, can be presentedin addition to producing the pseudo-colored image to the user.In order to present the MHSI effectively, a display method was developedthat has the potential to convey a 2-dimensional index, and convey thevalues for both the oxy and deoxyhemoglobin coefficients independently.The method of displaying our index uses a color scale, in one iterationthis ranges from purple values (high) to brown values (low) to indicatethe concentration of oxyhemoglobin in the tissue, and a brightnessscale, ranging from very bright (high) to faded (low) associated withthe tissue concentration of deoxyhemoglobin. FIG. 3 summarizes thedisplay of the MHSI, showing a schematic diagram explaining thescenarios of low and high oxy and deoxy hemoglobin coefficients as wellas a color scale that indicates a color plat that shows the verticalcolor scale and the horizontal brightness scale. By measuring an MHSIwhere the oxyhemoglobin component is high and the deoxyhemoglobincomponent is low (upper left hand corner of FIG. 3), it could beconcluded that that particular area of tissue has adequate perfusion andoxygenation, and is able to satisfy its metabolic needs with the oxygenthat is being delivered. That this tissue has the lowest level of riskfor ulceration and the highest probability of healing. If tissuedemonstrates a low oxyhemoglobin level in addition to a lowdeoxyhemoglobin level (lower left corner of FIG. 3), this would implythat the tissue was receiving low total volume of blood. If tissuedemonstrates a low oxyhemoglobin level in addition to a highdeoxyhemoglobin level (lower right corner of FIG. 3) this would implythat the tissue has metabolic requirements exceeding available oxygendelivery. In both of these regions there is expected to be a higher riskof ulceration or difficulties with wound healing. If the tissue has ahigh oxyhemoglobin coefficient and also has a high deoxyhemoglobincontent, (lower left corner of FIG. 3) this tissue was receiving alarger total volume of blood, and that the oxygen extracted from theblood stream was adequate to support tissue metabolism. This could beindicative of inflammation. Our technique will uniquely permitdiscrimination between each of these disparate physiologic conditions.For example, if the value is faded purple (upper left hand quadrant) thetissue has very high oxygenation, as discussed above, and is very likelyto heal. The color map (right) gives an indication of how the MHSI wouldbe represented in an image format.

Described here is hyperspectral imaging for use in the peripheralvascular and diabetes clinic, designed both to be mobile for ease of useand to facilitate the most accurate data collection possible for thisproject. This system provides fast and precise measurement ofreflectance spectra, and is characterized by high spatial and spectralresolution, and the ability to process spectral data in real time. Ithas been equipped with a turn-key software interface for the user.Proprietary image registration software insures image stability whenmeasuring spectra of animated objects. The system does not rely onexternal illumination, rather it contains very efficient internalvisible (and NIR in certain versions) light sources, which allow toachieve high signal to noise ratios in measured data without puttingnoticeable heat load on a biological subject (variations in skintemperature during acquisition are on the order of 0.1 C).

All MHSI data were corrected for background and uneven illumination, andnormalized by the integration time. The data were ratioed to thereflectance of a calibration standard, and negative decimal logarithmwas taken to obtain the absorption data. Images at all wavelengths wereco-registered using proprietary software developed by HYPERMED to ensurethat each pixel represents the same point on the skin throughout allwavelengths. The spectra were then deconvolved into fourlinearly-independent spectral components with coefficients representingthe amount of hemoglobin (both oxyHb and deoxyHb) in the observed skin.Typically two numbers are presented x/y wherein x represents oxyHb and yis deoxyHb. The values for x and y can be taken from a single pixel orfrom a ROI defined by the user. In addition the hemoglobin oxygensaturation (S_(HSI)O₂), x/(x+y), can be presented for a pixel or a ROI.FIG. 4 shows examples of tissue with low and high oxyHb and deoxyHbvalues corresponding to tissue at risk of ulceration and a wound that islikely to heal, respectively. Another way of presenting linearlyindependent variables is through their sum and difference: (x+y) and(x−y). The first would be THb, and the second would “hint” on oxygenextraction—which indicates the kind of Hb that is predominant at thesite.

Data in the following table represent typical oxyHb, deoxyHb, andS_(HSI)O₂ values for two body positions, forearm and foot, and forvarious stages of diabetes: nondiabetics, diabetics without peripheralneuropathy, and diabetics with peripheral neuropathy. In general, thevalue for oxyHb and S_(HSI)O₂ are lower in the feet of diabetic subjectswith neuropathy compared to the other two groups, a group at high riskfor developing foot ulcers. In addition, the values for oxyHb, deoxyHb,and S_(HSI)O₂ depend on body location, that once calibrated can beaccounted for by the diagnostic module.

MHSI oximetry values at baseline (prior to iontophoresis ofacetylcholine) Site Group (N) Oxy Deoxy S_(HSI)O₂ (%) Forearm Control(21) 29 ± 7*  41 ± 16 42 ± 17**  Diabetic Non- 20 ± 5   44 ± 10 32 ±8**   Neuropathic (36) Diabetic 19 ± 7   49 ± 10 28 ± 8**   Neuropathic(51) Dorsum of foot Control (21) 25 ± 13  44 ± 18 38 ± 22   DiabeticNon- 24 ± 9   41 ± 11 37 ± 12   Neuropathic (36) Diabetic 19 ± 9*** 45 ±13 30 ± 12**** Neuropathic (51) *p < 0.0001 compared to diabetics withand without neuropathy **p < 0.0001 for all three groups ***p < 0.025when compared to control and nonneoropathic ****p < 0.027 when comparedto control and nonneoropathic

In summarizing these data, MHSI provides relevant physiologicalinformation at the systemic, regional and local levels. Forearm datameasures systemic microvasculature changes since the forearm is notaffected by macrovasculature or somatic neuropathy as found in the lowerextremities. Dorsal foot measurements are indicative of microvascularand macrovascular effects including atherosclerotic changes occurring inlarge vessels exacerbated by diabetes. MHSI data from the right and leftlower extremity can be compared to help differentiate the stages of thedamage. Finally, MHSI can be used to find local information that can beassociated to the risk of developing a foot ulcer of the progression ofdisease by examining the area around an ulcer.

MHSI can not only be used for determining risk of foot ulceration, butalso for determining systemic progression of diabetic microvasculardisease. In one embodiment this is determined by mean oxyHb, deoxyHband/or other values for a region of interest. In another embodiment thisis determined by heterogeneity of oxyHb, deoxyHb and/or other values fora region of interest. In another embodiment this is determined by thepatterning of oxyHb, deoxyHb and/or other values for a region ofinterest. In other embodiments this is determined by changes over agiven time period within a measurement session (between 1 picosecond andone hour preferably between 100 microseconds and 10 minutes and morepreferably between 100 microseconds and 15 seconds) and in the meanvalues or patterns of oxyHb, deoxyHb and/or other values for a region ofinterest. These measurements can be used to determine a diabetesprogression index (DPI). Alternatively, a DPI can be calculated bycomparing a MHSI value or set of values from a single point in time withanother point in time (preferably 1 month to two years, more preferably2 months to one year and most preferably 3-6 months)

Using the active stimulus of acetylcholine as a vasodilator, and theknown effects of this on LD measurements data was derived in whichchanges in MHSI could be observed under known alterations in physiologyand compare these with baseline images and with LD data (FIG. 5). Usingdata collected from diabetic patients, as well as previous data fromhuman shock studies and iontophoresis studies, an algorithm was derivedthat clearly discriminates regions vasodilated by iontophoresis and alsodiscriminates ulcer from non ulcer with a proprietary formula thatincludes terms for oxyHb and deoxyHb. This was further developed as aHyperspectral Microvascular Index (HMI), which is a metric of tissuephysiology and have explored the use of the MHSI in evaluating tissue ofthe foot. In circumstances when an ulcer has been present, tissue wasexamined within the ulcer, directly adjacent to the ulcer, surroundingthe ulcer and at various other regions of the foot.

With the aid of MHSI, a quantitative metric is demonstrated with superbseparation between ulcerated or wounded and non-ulcerated or woundedtissue. Areas of different tissue metabolism can be seen with 60 micron(20-120) spatial resolution. Regions with an increased MHSI associatedwith the margins of the ulcer can be seen which correlate toinflammation (and/or infection). Areas of decreased MHSI can be seen inother areas which from previous work in ischemia is considered to betissue at risk for non-healing, ulcer extension, or primary ulceration.These data validate the capability of our measurement system to have theresolution and appropriate range to quantitatively assess differentareas of tissue metabolism on both dorsal and plantar foot surfaces aswell as skin on other body areas or other tissues visible throughendoscopic techniques or at the time of open surgery of the foot, leg,arm or any other body part including internal organs at laparoscopy orthe retina at retinoscopy. The invention provides the capability toperform this quantitative assessment on tissue that demonstrates novisible differences on clinical examination to the skilled examiner.

MSHI images have the ability to differentiate between regions of tissueassociated with a present foot ulcer on the basis of biomarkers such asthe oxyHb and deoxyHb coefficients. FIG. 6 shows an ulcer on the sole ofthe foot of a type 1 diabetic patient (ulcer 1). From the visible imageon the left, little distinguishes one area of the ulcer from another.However when looking at the image with the MHSI, there is obviousdiscriminatory power between the state of tissue seen in the purpleoval, which is likely to heal, and that surrounded by the black oval,which is tissue at risk for further ulceration. It is important to notethat the skin on the sole of this patient's feet is highly calloused,with a thick stratum corneum, but one is still able to differentiatetissue based on its spectral signatures. Given that the sole of the footis often the site of the thickest stratum corneum on the body, thedevice works on all naturally or surgically exposed tissue or tissueotherwise visualized with laparoscopy, endoscopy, retinoscopy or othervisualization techniques. Ulcer 2 was located on the dorsal surface ofthe foot, on the patient's big toe (FIG. 6). These images further showthe ability to differentiate between tissue at risk and tissue likely toheal. Additionally, tissue surrounding a fungal infection on thepatient's middle toe (bottom right-hand corner of the image) has an MHSIthat can demonstrate inflamed or infected tissue.

In addition to the differentiation of local tissue, tissue can beexamined for gross features indicative of global risks of complications,such as poor perfusion or the inability of the microcirculation to reactand compensate in tissue. In another embodiment, iontophoreticapplication of the vasodilator acetylcholine (ACH) or nitroprusside wasused to stimulate the vasodilation of the microvasculature on the dorsalsurface of the foot and on the forearm of the patients and measured thereaction with MHSI (FIG. 7).

There is potential for hyperspectral imaging in diagnosing globalmicrocirculatory insufficiencies and impacting other complications ofdiabetes associated with the microvasculature besides foot ulcers. InFIG. 7, hyperspectral measurements from the feet of four patients, withthe first two columns of images showing the MHSI of the soles of bothfeet, and the second two columns showing images of the dorsal surface ofboth feet after the application of ACH via iontophoresis. In the firstthree patients, an MHSI is seen that is much healthier than that of thefourth patient. Consequently, the fourth patient had a foot ulcer at thetime of this study and has a previous history of ulceration. While thecontrast between the data from the soles in these patients is striking,there is complementary information in the data from the microvascularresponse shown in the two columns on the right. Note that the firstthree patients all have MHSI scores that contain purple information inresponse to vasodilation, while the fourth patient shows what would beconsidered an MHSF that was indicative of tissue that was at risk.Microcirculatory changes associated with the progression of diabetes canalso be modified by different treatment and therapeutic regimens andwith the overlay of other systemic diseases (such as congestive heartfailure or hypertension) or treatments or therapies for systemicdiseases.

To analyze the ulcer data further, ulcer images were divided into 25concentric circles 1 mm apart and 8 pie segments forming 200 sectors perulcer (FIG. 8). A radial profile analysis was undertaken where the ulcercenter was defined at the first visit, and registered images fromsubsequent visits to this. OxyHb, deoxyHb, total-hemoglobin and O₂Satwere calculated for each sector.

Each radial pie segment was evaluated for signs of healing, nonhealingor progression in subsequent visits. MHSI measurements and clinicalhealing results were compared. MHSI algorithms were developed toidentify changes associated with ulcer healing, nonhealing andprogression. A primary endpoint evaluated the specific sectors of tissuearound an ulcer that would heal, not heal or progress. The groupestimates for oxyHb, deoxyHb, and O₂Sat are given in the following tableusing a linear mixed effects regression model. Significant differenceswere seen for healing for the oxyHb and deoxyHb values. Patients who didnot heal also demonstrated increased heterogeneity in distant foot andin arm measurements. For the 21 ulcers studied, the algorithm predicted6 of 7 ulcers that did not heal and 10 of 14 ulcers that healed.Conclusion: MHSI identifies microvascular abnormalities in the diabeticfoot and provides early information assist in managing foot ulcerationand predict outcomes in patients with diabetes.

Group Estimates (±SEM) MHSI Not Healing Healing p-value OxyHb 36.4 ± 2.251.9 ± 1.8 <.0001 DeoxyHb 34.2 ± 1.9 47.8 ± 1.6 <.0001 S_(HSI)O₂  0.51 ±0.01  0.51 ± 0.01 0.8646

MHSI is used to monitor angiogenesis during wound healing. An example ofwound healing in a diabetic rabbit wound model shows that during thehealing process, images of oxyHb and deoxyHb show patterns that changein shape, area, and amplitude with time. Similar patterns were noted inexperimental models of shock, but the changes observed for shockoccurred on a shorter time scale; minutes rather than several days asthe wound heals. The rabbit's ears were observed at days 1, 2, 5, and10. MHSI is ideally suited for characterization of the localheterogeneity in oxyHb and deoxyHb and their spatial changes with time.For example, the zone of hyperemia surrounding a wound as measured bythe oxyHb coefficient decreases with time in wounds that heal (FIG. 9).The color image (a), reconstructed from MHSI data, shows a part of theobserved area 50-by-40 mm, recorded at the baseline on day 1. The blackrings denote location of a future wound. The pseudocolor image (b),obtained as a result of hyperspectral processing, shows distribution ofthe oxyHb and deoxyHb in the underlying tissue at the same time. Thecolor hue represents apparent oxyHb concentrations, whereas colorsaturation (from fade to bright) represents apparent deoxyHbconcentrations. Both, oxyHb and deoxyHb vary predominantly between 40and 90 MHSI units (color bar to the right). The remaining images to theright show change in a region of interest 17-by-17-mm (black box in (a)and (b)) over 10 days. At day 2, the oxy concentrations increasedsignificantly in the area as far as 10 mm away from the wound border. Byday 5, the increase in oxygenation became more local (purple area,shrunken to about 5 mm) and new microvasculature formed to feed the areain need (red fork-like vessels in the right top corners appearing indays 5 and 10 images). By the 10^(th) day, the area of increased oxyHbhas not changed much, but the peak in oxy amplitude decreased,suggesting a period of steady healing.

As depicted in FIG. 10, 50-micron resolution images of a rabbit's earwere taken with MHSI over a ten day period. In FIG. 10(a), the colorimage was reconstructed from MHSI data, showing a party of the observedarea 50-by-40 mm, recorded at the baseline on day 1. The pseudo-image(b) was obtained as a result of hyperspectral processing, showing adistribution of the oxygenated (oxy) and deoxygenated (deoxy) hemoglobinin the underlying tissue at the same time.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications. U.S. and foreign patents, and patent andprovisional applications, and all publications and documents citedherein for any reason, are specifically and entirely incorporated byreference. It is intended that the specification and examples beconsidered exemplary only.

LITERATURE CITED

-   1. International Diabetes Federation. Information on Global Diabetes    Prevalence. In: http://www.eatlas.idf.org/Prevalence/; 2004.-   2. American Diabetes Association.    http://www.diabetes.org-diabetes/complications.jsp. 2005.-   3. Brearley S, Shearman C P, Simms M H. Peripheral pulse palpation:    an unreliable physical sign. Ann R Coll Surg Engl 1992;    74(3):169-71.-   4. Frykberg R G, Layery L A, Pham H, Harvey C, Harkless L, Veves A.    Role of neuropathy and high foot pressures in diabetic foot    ulceration. Diabetes Care 1998; 21(10):1714-9.-   5. Sumpio B E. Foot ulcers. N Engl J Med 2000; 343(11):787-93.-   6. Young M J, Breddy J L, Veves A, Boulton A J. The prediction of    diabetic neuropathic foot ulceration using vibration perception    thresholds. A prospective study. Diabetes Care 1994; 17(6):557-60.-   7. Cavanagh P, Young M, Adams J, al. e. Correlates of structure and    function in neuropathic diabetic feet. Diabetologia 1991; 34(Suppl    2):A39 (abstract).-   8. Layery L A, Armstrong D G, Vela S A, Quebedeaux T L, Fleischli    J G. Practical criteria for screening patients at high risk for    diabetic foot ulceration. Arch Intern Med 1998; 158(2):157-62.-   9. McMillan D E. Development of vascular complications in diabetes.    Vasc Med 1997; 2(2):132-42.-   10. Novo S. Classification, epidemiology, risk factors, and natural    history of peripheral arterial disease. Diabetes Obes Metab 2002; 4    Suppl 2:S1-6.-   11. Hittel N, Donnelly R. Treating peripheral arterial disease in    patients with diabetes. Diabetes Obes Metab 2002; 4 Suppl 2:S26-31.-   12. Pecoraro R E, Reiber G E, Burgess E M. Pathways to diabetic limb    amputation. Basis for prevention. Diabetes Care 1990; 13(5):513-21.-   13. Reiber G E, Boyko E J, Smith D C. Lower extremity foot ulcers    and amputations in diabetes. In: Harris, Cowie, Stern, Boyko E J,    Reiber G E, Bennet, editors. Diabetes in America. 2nd ed.    Washington, D.C.: US Government Printing Office; 1995. p. 402-428.-   14. Palumbo P J, Melton L J. Peripheral vascular disease and    diabetes. In: Harris, Hamman, editors. Diabetes in America. 1st ed.    Washington, D.C.: US Government Printing Office; 1985.-   15. Frykberg R G, Armstrong D G, Giurini J, Edwards A, Kravette M,    Kravitz S, et al. Diabetic foot disorders: a clinical practice    guideline. American College of Foot and Ankle Surgeons. J Foot Ankle    Surg 2000; 39(5 Suppl):S1-60.-   16. Ramsey S D, Newton K, Blough D, McCulloch D K, Sandhu N, Reiber    G E, et al. Incidence, outcomes, and cost of foot ulcers in patients    with diabetes. Diabetes Care 1999; 22(3):382-7.-   17. Harrington C, Zagari M J, Corea J, Klitenic J. A cost analysis    of diabetic lower-extremity ulcers. Diabetes Care 2000;    23(9):1333-8.-   18. Caputo G M, Cavanagh P R, Ulbrecht J S, Gibbons G W, Karchmer    A W. Assessment and management of foot disease in patients with    diabetes. N Engl J Med 1994; 331(13):854-60.-   19. Layery L, Gazewood J D. Assessing the feet of patients with    diabetes. J Fam Pract 2000; 49(11 Suppl):59-16.-   20. Frykberg R G. Diabetic foot ulcers: pathogenesis and management.    Am Fam Physician 2002; 66(9):1655-62.-   21. Sykes M T, Godsey J B. Vascular evaluation of the problem    diabetic foot. Clin Podiatr Med Surg 1998; 15(1):49-83.-   22. Treado P J, Morris M D. Infrared and Raman spectroscopic    imaging. Appl Spectrosc Rev 1994; 29:1-38.-   23. Riaza A, Strobl P, Muller A, Beisl U, Hausold A. Spectral    mapping of rock weathering degrees on granite using hyperspectral    DAIS 7915 Spectrometer Data. Internl J Applied Earth Observation and    Geoinformation Special issue: Applications of imaging spectroscopy    2001; 3-4:345-354.-   24. Thenkabail P S, Smith R B, De Pauw E. Hyperspectral vegetation    indices and their relationships with agricultural crop    characteristics. Remote Sens Environ 2000; 71(REMOTE SENS    ENVIRON):158-182.-   25. Colarusso P, Kidder L H, Levin I W, et al. Infrared    spectroscopic imaging: from planetary to cellular systems. Appl    Spectrosc 1998; 52:106A-120A.-   26. HyperMed, Inc., Issued U.S. Pat. Nos. 6,937,885; 6,810,279,    6,741,884; 6,640,130; and 6,640,132; and others pending.-   27. Freeman J E, Shi Y, Panasyuk S V, Rogers A E. Medical    hyperspectral imaging (MHSI) of 1,2-dimethylbenz(a)-anthracene    (DMBA)-induced breast tumors in rats. Poster #1001. In: 27th Annual    San Antonio Breast Cancer Symposium: 2004; San Antonio, Tex.: Breast    Cancer Research and Treatment; 2004. p. S51.-   28. Carlson K A, Jahr J S. A historical overview and update on pulse    oximetry. Anesthesiol Rev 1993; 20:173-181.-   29. Zuzak K J, Schaeberle M D, Gladwin M T, et al. Noninvasive    determination of spatially resolved and time-resolved tissue    perfusion in humans during nitric oxide inhibition and inhalation by    use of a visible-reflectance hyperspectral imaging technique.    Circulation 2001; 104:2905-2910.-   30. Afromowitz M A, Callis J B, Heimbach D M, DeSoto L A, Norton    M K. Multispectral imaging of burn wounds: a new clinical instrument    for evaluating burn depth. IEEE Trans Biomed Eng 1988;    35(10):842-50.-   31. Sowa M G, Leonardi L, Payette J R, Fish J S, Mantsch H H. Near    infrared spectroscopic assessment of hemodynamic changes in the    early post-burn period. Burns 2001; 27:241-9. Burns 2001;    27:241-249.-   32. Dinh T, Panasyuk S, Freeman J, Panasyuk A A, Lew R, Brand D, et    al. Medical hyperspectral imaging (MHSI) evaluation of    microcirculatory changes to predict clinical outcomes: Application    to diabetic foot ulcers. Society of Vascular Medicine and Biology    17th Annual Scientific Session 2006:(Abstract submitted).-   33. Dinh T, Panasyuk S V, Jiang C, Freeman J, Panasyuk A A, Nerney    M, et al. The use of medical hyperspectral imaging (MHSI) to    evaluate microcirculatory changes in diabetic foot ulcers and    predict clinical outcomes. American Diabetes Association 66th    Scientific Session 2006:(abstract accepted).-   34. Dinh T, Panasyuk S V, Tracey B H, Khaodhiar L, Lyons T E,    Rosenblum B L, et al. The use of medical hyperspectral imaging    (MHSI) to identify patients at risk for developing diabetic foot    ulcers. Diabetes 2005; 54(S1):A270.-   35. Dinh T, Panasyuk S V, Tracey B H, Khaodhiar L, Lyons T E,    Rosenblum B L, et al. The use of medical hyperspectral imaging    (MHSI) to identify patients at risk for developing diabetic foot    ulcers. American Diabetes Association 65th annual session    2005:Poster: 1106-P, June.-   36. Greenman R I, Panasyuk S, Wang X, Lyons T E, Dinh T, Longorio L,    et al. Early changes in the skin microcirculation and muscle    metabolism of the diabetic foot. Lancet 2005; 366: 1711-1718. Lancet    2005; 366:1711-1718.-   37. Mansfield J R, Lew R A, Lewis E N, Freeman J E, Kellicut D,    Sidawy A. Hyperspectral imaging of diabetic foot conditions. In:    Eastern Analytical Society Meeting; 2003; Somerset, N.J.; 2003.-   38. Freeman J E, Panasyuk S V, Hopmeier M J, Lew R A, Batchinski A    I, Cancio L C. The evaluation of new methods of hyperspectral image    analysis for the diagnosis of hemorrhagic shock, Poster #17. In:    American Association for the Surgery of Trauma; 2005; 2005.-   39. Gillies R, Freeman J E, Cancio L C, Brand D, Hopmeier M,    Mansfield J R. Systemic effects of shock and resuscitation monitored    by visible hyperspectral imaging. Diabetes Technol Therapeut 2003;    5(5):847-855.-   40. Panasyuk S V, Freeman J E, Cooke W I, Hopmeier M J, Convertino    V A. Initial demonstration in human subjects of medical    hyperspectral imaging (MHSI) as a novel stand-off non-invasive    method for diagnosing and measuring hemodynamic compromise, Poster    #18. In: American Association for the Surgery of Trauma; 2005; 2005.-   41. Payette J R, Sowa M G, Germscheid S L, Stranc M F, Abdulrauf B,    Mantsch H H. Noninvasive diagnostics: predicting flap viability with    near-IR spectroscopy and imaging. Am Clinical Laboratory 1999;    18:4-6.-   42. Armstrong D G, Layery L A. Predicting neuropathic ulceration    with infrared dermal thermometry. J Am Podiatr Med Assoc 1997;    87(7):336-7.-   43. Beckert S, Witte M B, Konigsrainer A, Coerper S. The Impact of    the Micro-Lightguide O2C for the Quantification of Tissue Ischemia    in Diabetic Foot Ulcers. Diabetes Care 2004; 27(12):2863-2867.-   44. Rajbhandari S M, Harris N D, Tesfaye S, Ward J D. Early    identification of diabetic foot ulcers that may require intervention    using the micro lightguide spectrophotometer. Diabetes Care 1999;    22(8):1292-5.-   45. Khan F, Newton D J. Laser Doppler imaging in the investigation    of lower limb wounds. Int J Low Extrem Wounds 2003; 2(2):74-86.-   46. Sheffield P J, Fife C E, Smith A P S. Laser Doppler Flowmetry.    In: Wound Care Practice. Flagstaff, Ariz.: Best Publishing Company;    2004.-   47. Wardlaw J M, Dennis M S, Warlow C P, Sandercock P A. Imaging    appearance of the symptomatic perforating artery in patients with    lacunar infarction: occlusion or other vascular pathology? Ann    Neurol 2001; 50(2):208-15.-   48. Zimny S, Dessel F, Ehren M, Pfohl M, Schatz H. Early detection    of microcirculatory impairment in diabetic patients with foot at    risk. Diabetes Care 2001; 24(10):1810-4.

The invention claimed is:
 1. An apparatus comprising: a) a spectralimager that is configured to resolve light obtained from a region ofinterest of a subject into a plurality of component spectral bands,wherein the spectral imager comprises a lens in optical connection withan image sensor; and b) a computer system electrically coupled to thespectral imager and comprising a memory and a processor, wherein thememory stores instructions for (i) constructing a color photo image byconcatenating three planes from a hyperspectral cube constructed fromthe plurality of component spectral bands; (ii) constructing a pseudocolor image based on the plurality of component spectral bands; and(iii) combining the pseudo color image with the color photo image toform a composite image, wherein the constructing of the pseudo colorimage comprises: defining a color hue plane that represents a firstcharacteristic in the group consisting of (a) an oxyhemoglobinconcentration, (b) a deoxyhemoglobin concentration and (c) amathematical combination of the oxyhemoglobin concentration and thedeoxyhemoglobin concentration; defining a color saturation plane thatrepresents a second characteristic in the group consisting of (a) theoxyhemoglobin concentration, (b) the deoxyhemoglobin concentration and(c) a mathematical combination of the oxyhemoglobin concentration andthe deoxyhemoglobin concentration, wherein the second characteristic isdifferent than the first characteristic; and defining a color intensityplane that represents reflectance in a region that is about 450 nm toabout 580 nm.
 2. The apparatus of claim 1, wherein a wavelength range ofeach respective component spectral band in the plurality of componentspectral bands is adjacent to the wavelength range of another componentspectral band in the plurality of component spectral bands.
 3. Theapparatus of claim 1, wherein each respective component spectral band inthe plurality of spectral bands has a bandwidth of less than 50 nm. 4.The apparatus of claim 1, wherein the memory further stores instructionsfor displaying the composite image on a computer screen.
 5. Theapparatus of claim 1, wherein the region of interest of the subject is aportion of the skin of the subject.
 6. The apparatus of claim 1, whereinthe light obtained from the region of interest is in the visible range.7. The apparatus of claim 1, wherein the color photo image isconstructed by concatenating a first resolved component spectral band, asecond resolved component spectral band, and a third resolved componentspectral band, wherein: the first resolved component spectral bandcomprises a wavelength corresponding to red light; the second resolvedcomponent spectral band comprises a wavelength corresponding to greenlight; and the third resolved component spectral band comprises awavelength corresponding to blue light.
 8. The apparatus of claim 1,wherein the three planes concatenated from the hyperspectral cubeconsist of a first plane at a wavelength of 580 nm to 800 nm, a secondplane at a wavelength of 480-580 nm, and a third plane at a wavelengthof 350 nm to 490 nm.
 9. The apparatus of claim 1, wherein the spectralimager further comprises one or more polarizers, wherein the one or morepolarizers compile light entering the spectral imager into a plane ofpolarization before entering the image sensor.
 10. The apparatus ofclaim 1, wherein the lens comprises a lens assembly.
 11. The apparatusof claim 10, wherein the lens assembly comprises a first stage imagingoptic and a second stage imaging optic.
 12. The apparatus of claim 1,further comprising a display for displaying the composite image.
 13. Theapparatus of claim 1, wherein the computing system further comprises astorage device for storing the composite image.
 14. The apparatus ofclaim 1, further comprising a printer for printing the composite image.15. The apparatus of claim 1, further comprising a communication linkfor transferring the composite image to a remote device or computer. 16.The apparatus of claim 1, wherein each respective component spectralband in the plurality of component spectral bands has a bandwidth thatis between 10 nm and 40 nm.
 17. The apparatus of claim 1, wherein eachrespective component spectral band in the plurality of componentspectral bands has a bandwidth that is between 10 nm and 15 nm.
 18. Theapparatus of claim 1, wherein each respective component spectral band inthe plurality of component spectral bands has a bandwidth that isbetween 5 nm and 12 nm.
 19. The apparatus of claim 1, wherein theapparatus is a portable apparatus.
 20. The apparatus of claim 1, whereinthe region of interest is a tissue of the subject.
 21. The apparatus ofclaim 20, wherein the tissue comprises an ulcer, callus, intact skin,hematoma, or superficial blood vessel.